Week 5 – Results and Restarting

I completed the proliferation assay with RvD1 resolvin this week and have finished analyzing the results!

Before I share the results, I thought it might be fun to share what this week in the lab looked like and some of the road bumps I encountered:

On Monday (Day 7 of the assay), I looked at the cell plate under a microscope to make sure no contamination was present. Then, I prepared the AlamarBlue and media mixture and pipetted this into all of the wells. I was not paying close enough attention on Monday and accidentally doubled the volume of the mixture in the wells 😦 . I should have triple checked the volume on the pipet before releasing it, but this is only a minor setback (I just won’t use Day 7 data when calculating the results). After adding the mixture, the cell plate must be incubated for 4 hours before it can read by the plate reader. Cell-Proliferation-Assay-Kits-32.jpg After I read the plate, I changed the media of the cells and added a new round of the resolvin drug. I have been repeating these steps for past two weeks and today was Day 11, the last day of the assay. 

Today was also the first day I was able to share what I have been working on during the weekly lab meetings. At first, it was honestly intimidating to present in front of some of the most qualified and knowledgeable people I have met. But I have realized that sharing is apart of the learning experience and leads to discussions that involve the exchanging of ideas and aids in progress.

Now to share the results:

For this assay the results are determined through a percent reduction (% red.) calculation. For our purposes, % reduction is equivalent to the percent of cell growth or proliferation. Here is the template of my cell plate:

Screen Shot 2018-03-16 at 12.54.38 PM.png

I had two control groups with no resolvin, resolvin (RvD1) at different nM concentrations, a media only control, and a Vitamin C control (the 100% reduced form of Alamarblue).

And here are the results from the % reduction calculation:

Screen Shot 2018-03-16 at 1.37.15 PM.png

Day 1: Purple, Day 3: Blue, Day 9: Green

This chart sums up my results from Day 1 through Day 9, with the omission of Day 7 data (the unfortunate day I doubled the AlamarBlue volume…). I did expect to see a bigger difference between the control and the 100 nM concentration of RvD1 in the proliferation of the cells. All the bars with RvD1 should be shorter than the control group bars (CTR, 0 nM of resolvin). This is only true for Day 9, but not Day 3. Thus, the resolvin did not significantly slow down the proliferation of the vascular smooth cells as intended. To learn more about why I expected the cells to grow less when treated with resolvin be sure to read last week’s post.

What now? I am going to repeat this assay next week with different controls that will be more accurate. I might also switch to the MTT assay for more precise results.

The MTT cell proliferation assay measures cell proliferation rate and reduction in cell viability.

My advisor told me to keep in mind that an AlamarBlue assay assumes that the metabolism of all the cells are constant throughout the experiment, which is never the case. He also said that I should create a growth curve with the upcoming results. I will keep all of this in mind while amending this experiment next week!

I have also decided to share one risk factor of coronary heart disease or plaque buildup in arteries at the end of every blog post. The more literature I read about the resolution of vascular inflammation after vessel wall injury has me wondering how vessel wall injury can be prevented in the first place. If a stent is never inserted in your artery, restenosis is a complication you never have to worry about!

This week’s risk factor: Smoking

Smoking can contribute to inflammation in the blood vessels. This inflammation can lead to plaque buildup in the arteries which supply your heart muscle with oxygen-rich blood. Smoking also thickens our blood, making it harder for our blood to carry oxygen. Studies have found that people who smoke are more likely to develop heart disease and suffer from a heart attack.

smoke-and-heart-diseaseTo learn more about the effects of smoking on your heart: Smoking and Your Heart

Thank you for reading and hope you will join me next week!

5 thoughts on “Week 5 – Results and Restarting

  1. Excellent blog post Amrita, I love your idea of sharing risk factors with your peers. If you educate yourself and peers to reduce or eliminate risk factors that cause inflammation of blood vessels, you have already made a positive impact.

    I am curious to see what the MTT assay shows. Carry on the good work

    Liked by 1 person

  2. Pingback: Week 11 – A New Drug and Freezer | The Effectiveness of Drug-Eluting Stents

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s